Anti-infective binding proteins that bind AIP2

ABSTRACT

There is disclosed compositions and methods relating to or derived from anti-AIP2 antibodies. More specifically, there is disclosed fully human antibodies that bind AIP2, AIP2-binding fragments and derivatives of such antibodies, and AIP2-binding polypeptides comprising such fragments. Further still, there is disclosed nucleic acids encoding such antibodies, antibody fragments and derivatives and polypeptides, cells comprising such polynucleotides, methods of making such antibodies, antibody fragments and derivatives and polypeptides, and methods of using such antibodies, antibody fragments and derivatives and polypeptides, including methods of treating or diagnosing subjects having AIP2 related disorders or conditions. There is also disclosed a method to treat  S. aureus  infections by administering an anti-AIP2 antibody described herein.

CROSS REFERENCE TO RELATED APPLICATION

This patent application claims priority from U.S. Provisional Patentapplication 61/719,103 filed 26 Oct. 2012.

The present invention was made with partial support of NIH grant1R42AI098182. The Federal Government has certain rights in thisinvention.

TECHNICAL FIELD

The present disclosure provides compositions and methods relating to orderived from anti-auto-inducing peptide (AIP) 2 antibodies. Morespecifically, the present disclosure provides human antibodies that bindAIP2, AIP2-binding fragments and derivatives of such antibodies, andAIP2-binding polypeptides comprising such fragments. Further still, thepresent disclosure provides nucleic acids encoding such antibodies,antibody fragments and derivatives and polypeptides, cells comprisingsuch polynucleotides, methods of making such antibodies, antibodyfragments and derivatives and polypeptides, and methods of using suchantibodies, antibody fragments and derivatives and polypeptides,including methods of treating or diagnosing subjects having AIP2 relatedbacterial infections, including infections caused by Staphylococcusaureus in general and particularly methicillin-resistant Staphylococcusaureus (MRSA).

BACKGROUND

Ever since it was first discovered by Sir Alexander Ogston in 1880,Staphylococcus aureus has been regarded as a serious threat to humanhealth, capable of causing a multitude of infections. The rise ofantibiotic-resistant strains in the 1960s and 1970s, particularlymethicillin-resistant S. aureus (MRSA), has created additionaltherapeutic challenges. Currently, MRSA strains account for >50% of allS. aureus isolates causing clinical disease in the US. This is a muchhigher percentage compared to other countries, such as France at 14.5%and the Netherlands at 3.1%. In a review of 31 observational studiesfrom Western Europe, the authors found that the percentage of MRSA amongS. aureus clinical isolates ranged between 5% and 54%, but was limitedby the different methodologies used in the studies.

Methicillin-resistant Staphylococcus aureus (MRSA) is a bacteriumresponsible for several difficult-to-treat infections in humans. It isalso called multidrug-resistant Staphylococcus aureus andoxacillin-resistant Staphylococcus aureus (ORSA). MRSA is any strain ofStaphylococcus aureus that has developed resistance to beta-lactamantibiotics, which include the penicillins (such as methicillin,dicloxam, nafcillin and oxacillin) and the cephalosporins. Strainsunable to resist these antibiotics are classified asmethicillin-sensitive Staphylococcus aureus (MSSA). The development ofsuch resistance does not cause the organism to be more intrinsicallyvirulent than strains of Staphylococcus aureus that have no antibioticresistance, but resistance does make MRSA infection more difficult totreat with standard types of antibiotics and thus more dangerous.

MRSA is especially troublesome in hospitals, prisons, schools, andnursing homes, where patients with open wounds, invasive devices, andweakened immune systems are at greater risk of infection than thegeneral public.

MRSA strains are prevalent bacterial pathogens that cause both healthcare- and community-associated infections. Increasing resistance tocommonly prescribed antibiotics has made MRSA a serious threat to publichealth throughout the world. The USA300 strain of MRSA has beenresponsible for an epidemic of community-associated infections in theUS, mostly involving skin and soft tissue but also more serious invasivesyndromes such as pneumonia, severe sepsis and endocarditis. MRSAstrains are particularly serious and potentially lethal pathogens thatpossess virulence mechanisms including toxins, adhesins, enzymes andimmunomodulators. One of these is Panton-Valentine leukocidin (PVL), atoxin associated with abscess formation and severe necrotizingpneumonia.

Initially, MRSA strains afflicted hospitalized patients and those withchronic illnesses. The 1990s saw the emergence of community-associatedMRSA (CA-MRSA) strains that primarily caused skin and soft tissueinfections (SSTIs) in otherwise healthy individuals, often children.These strains quickly led to an epidemic of CA-MRSA infections includingsome with severe consequences, for example, community-acquired pneumoniawith high mortality rates. The high prevalence of CA-MRSA amonginfecting MRSA strains in the US is mostly due to the Panton-Valentineleukocidin (PVL)-positive USA300 clone, while in Europe the predominantstrain of CA-MRSA is a PVL-positive ST80 clone. A mathematical modelpredicted that CA-MRSA will become the dominant MRSA strain in hospitalsbecause of the expanding community reservoir, CA-MRSA strains are morefit (higher replicative capacity) than hospital-associated types andthat CA-MRSA infections will become increasingly severe (D'Agata et al.,Clin. Infect. Dis. 48, 274-284, 2009).

Agents directed against the virulence mechanisms of MRSA strains wouldhave several advantages compared to antibiotics. First, there would beno selective pressure exerted on other nonpathogenic, commensalbacteria. Second, the associated toxicities of antibiotics (e.g.allergic reactions, nephrotoxicity and Clostridium difficile infection)may be avoided. Third, limiting antibiotics may decrease the developmentof drug-resistant bacteria. Combining antivirulence therapies withtraditional antibiotics has the potential to change the paradigm of howMRSA infections are managed. Since bacterial survival is not impacted bythe function of its virulence mechanisms, it is possible that resistanceto antivirulence therapy would be slow to develop. One potentialstrategy is to inhibit the agr operon. In vitro experiments have shownthat variants of autoinducing peptide (AIP) inhibit AgrC function. An invivo study demonstrated that administering AIP-2 concurrently with anagr type 1 strain reduced abscess formation (Wright et al., Proc. Natl.Acad. Sci. USA 102, 1691-1696, 2005). However, agr inhibitors canpromote biofilm formation, which could result in chronic S. aureusinfections (Beenken et al., PLoS ONE 5, e10790, 2010). Hence, furtherinvestigation on this approach is needed.

Another strategy for devices is the use of nanomaterials, defined asmaterials with at least one dimension less than 100 nm, to prevent theformation of biofilms (Taylor & Webster, Int. J. Nanomedicine 6,1463-1473, 2011). SilverPage lined urinary catheters and central venouscatheters are used in clinical practice to lower the risk of healthcare-associated infections (Raad et al., Antimicrob. Agents Chemother.56, 935-941, 2012). Decreasing the particle size of silver down to thenanometer range increases the surface area, which improves theantibacterial activity of the material (Taylor & Webster, Int. J.Nanomedicine 6, 1463-1473, 2011). Staphyloxanthin is a pigment of S.aureus that helps it resist reactive oxygen species such as thosereleased by neutrophils. Early steps in staphyloxanthin production aresimilar to those in cholesterol production. A human squalene synthaseinhibitor blocked staphyloxanthin biosynthesis in vitro, resulting innonpigmented bacteria that were more susceptible to killing by humanblood and clearance by the innate immune system in a mouse model (Liu etal., Science 319, 1391-1394, 2008). Statins were shown to enhance S.aureus clearance by phagocytes through production of antibacterialDNA-based extracellular traps by human and murine neutrophils,macrophages and monocytes (Chow et al., Cell Host Microbe 8, 445-454,2010).

For CA-MRSA infections, one specific target is PVL toxin, and antibodyagainst it is under investigation as a potential vaccine. However, in astudy on antibody levels against PVL in children with PVL-positive MRSAinfections, neutralizing antibody against PVL was not protective againstprimary or recurrent CA-MRSA skin infections (Hermos et al., Clin.Infect. Dis. 51, 1138-1146, 2010). Other investigators, using a murinemodel of dermonecrosis, evaluated an agonist of human C5a called EP67for its ability to induce host immunity against CA-MRSA (Sheen et al.,Vaccine 30, 9-13, 2011). EP67 was effective in limiting the infectionthrough the promotion of cytokine synthesis and neutrophil influx. Thispromising finding may warrant further investigation in humans.

Peptidoglycan (PG) comprises approximately 50% of the cell wall of S.aureus. A PG-based vaccine against S. aureus, A170PG, was shown to beprotective in a mouse model against several strains of MRSA includingA174, A175, A176 and RIMD31092 (Capparelli et al., PLoS ONE 6, e28377,2011). The protection correlated with increased survival and reducedcolonization and lasted at least 40 weeks. One caveat with this study isthat the mouse strain used does not closely mimic human infectionbecause mice do not have pre-existing antibodies to S. aureus. In June2011, Merck and Intercell announced the termination of phase II/IIIdevelopment of V170, a subunit vaccine containing the S. aureus antigenIsdB, which is a cell surface localized iron-regulated protein (Etz etal., Proc. Natl. Acad. Sci. USA 99, 6573-6578, 2002). Safety concernswere cited due to an increase in overall mortality and multi-organdysfunction in the vaccine recipients compared to those who receivedplacebo.

SUMMARY

The present disclosure provides a fully human antibody of an IgG classthat binds to an AIP2 epitope with a binding affinity of at least 10⁻⁶M,which has a heavy chain variable domain sequence that is at least 95%identical to the amino acid sequences selected from the group consistingof SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6,SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15,and combinations thereof, and that has a light chain variable domainsequence that is at least 95% identical to the amino acid sequenceconsisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8,SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, andcombinations thereof. Preferably, the fully human antibody has both aheavy chain and a light chain wherein the antibody has a heavychain/light chain variable domain sequence selected from the groupconsisting of SEQ ID NO. 1/SEQ ID NO. 2 (called A9 herein), SEQ ID NO.3/SEQ ID NO. 4 (called C7 herein), SEQ ID NO. 5/SEQ ID NO. 6 (called D3herein), SEQ ID NO. 7/SEQ ID NO. 8 (called E7 herein), SEQ ID NO. 9/SEQID NO. 10 (called F7 herein), SEQ ID NO. 11/SEQ ID NO. 12 (called G3herein), SEQ ID NO. 13/SEQ ID NO. 14 (called G4 herein), SEQ ID NO.15/SEQ ID NO. 16 (called H1 herein), and combinations thereof.

The present disclosure provides a Fab fully human antibody fragment,having a variable domain region from a heavy chain and a variable domainregion from a light chain, wherein the heavy chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ IDNO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 9, SEQ IDNO. 11, SEQ ID NO. 13, SEQ ID NO. 15, and combinations thereof, and thathas a light chain variable domain sequence that is at least 95%identical to the amino acid sequence consisting of SEQ ID NO. 2, SEQ IDNO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ IDNO. 14, SEQ ID NO. 16, and combinations thereof. Preferably, the fullyhuman antibody Fab fragment has both a heavy chain variable domainregion and a light chain variable domain region wherein the antibody hasa heavy chain/light chain variable domain sequence selected from thegroup consisting SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4,SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQID NO. 15/SEQ ID NO. 16, and combinations thereof.

The present disclosure provides a single chain human antibody, having avariable domain region from a heavy chain and a variable domain regionfrom a light chain and a peptide linker connection the heavy chain andlight chain variable domain regions, wherein the heavy chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 9,SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, and that has a light chainvariable domain sequence that is at least 95% identical to the aminoacid sequence consisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6,SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO.16, and combinations thereof. Preferably, the fully human single chainantibody has both a heavy chain variable domain region and a light chainvariable domain region, wherein the single chain fully human antibodyhas a heavy chain/light chain variable domain sequence selected from thegroup consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO.4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO.9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO.14, SEQ ID NO. 15/SEQ ID NO. 16, and combinations thereof.

The present disclosure further provides a method for treating orpreventing the S. aureus infection comprising administering an anti-AIP2polypeptide, wherein the fully human antibody has a heavy chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 9,SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, and combinations thereof,and that has a light chain variable domain sequence that is at least 95%identical to the amino acid consisting of SEQ ID NO. 2, SEQ ID NO. 4,SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14,SEQ ID NO. 16, and combinations thereof;

wherein the Fab fully human antibody fragment has the heavy chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 1, SEQID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ IDNO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, and combinationsthereof, and that has the light chain variable domain sequence that isat least 95% identical to the amino acid sequence consisting of SEQ IDNO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ IDNO. 12, SEQ ID NO. 14, SEQ ID NO. 16, and combinations thereof; and

wherein the single chain human antibody has the heavy chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 9,SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, and combinations thereof,and that has the light chain variable domain sequence that is at least95% identical to the amino acid sequence consisting of SEQ ID NO. 2, SEQID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQID NO. 14, SEQ ID NO. 16, and combinations thereof.

Preferably, the fully human antibody has both a heavy chain and a lightchain wherein the antibody has a heavy chain/light chain variable domainsequence selected from the group consisting of SEQ ID NO. 1/SEQ ID NO. 2(called A9 herein), SEQ ID NO. 3/SEQ ID NO. 4 (called C7 herein), SEQ IDNO. 5/SEQ ID NO. 6 (called D3 herein), SEQ ID NO. 7/SEQ ID NO. 8 (calledE7 herein), SEQ ID NO. 9/SEQ ID NO. 10 (called F7 herein), SEQ ID NO.11/SEQ ID NO. 12 (called G3 herein), SEQ ID NO. 13/SEQ ID NO. 14 (calledG4 herein), SEQ ID NO. 15/SEQ ID NO. 16 (called H1 herein), andcombinations thereof. Preferably, the fully human antibody Fab fragmenthas both a heavy chain variable domain region and a light chain variabledomain region wherein the antibody has a heavy chain/light chainvariable domain sequence selected from the group consisting of SEQ IDNO. 1/SEQ ID NO. 2 (called A9 herein), SEQ ID NO. 3/SEQ ID NO. 4 (calledC7 herein), SEQ ID NO. 5/SEQ ID NO. 6 (called D3 herein), SEQ ID NO.7/SEQ ID NO. 8 (called E7 herein), SEQ ID NO. 9/SEQ ID NO. 10 (called F7herein), SEQ ID NO. 11/SEQ ID NO. 12 (called G3 herein), SEQ ID NO.13/SEQ ID NO. 14 (called G4 herein), SEQ ID NO. 15/SEQ ID NO. 16 (calledH1 herein), and combinations thereof. Preferably, the fully human singlechain antibody has both a heavy chain variable domain region and a lightchain variable domain region, wherein the single chain fully humanantibody has a heavy chain/light chain variable domain sequence selectedfrom the group consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ IDNO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ IDNO. 14, SEQ ID NO. 15/SEQ ID NO. 16, and combinations thereof.

Preferably, the method for treating or preventing the S. aureusinfection is caused by a MRSA S. aureus bacteria.

DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that human anti-AIP mAbs C7 and E7 reduced mortality inmice infected intraperitoneally with USA100. *P≦0.0006 and 0.0008 versuscontrol IgG and E7 or C7 respectively as determined by Log Rank test

FIG. 2 shows that human anti-AIP mAbs C7 and E7 reduced abscess size inmice infected with USA100. *P≦0.01-0.04, E7 versus control on all daystested as determined by paired T-test.

FIG. 3 shows that human anti-AIP mAb E7 reduced mortality in miceinfected intraperitoneally with MSSA RN4850.

DETAILED DESCRIPTION

The present disclosure provides a fully human antibody of an IgG classthat binds to a AIP2 epitope with a binding affinity of 10⁻⁶M or less,that has a heavy chain variable domain sequence that is at least 95%identical to the amino acid sequences selected from the group consistingof SEQ ID NO. 1, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6,SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15,and combinations thereof, and that has a light chain variable domainsequence that is at least 95% identical to the amino acid sequenceconsisting of SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8,SEQ ID NO. 10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, andcombinations thereof. Preferably, the fully human antibody has both aheavy chain and a light chain wherein the antibody has a heavychain/light chain variable domain sequence selected from the groupconsisting of SEQ ID NO. 1/SEQ ID NO. 2 (called A9 herein), SEQ ID NO.3/SEQ ID NO. 4 (called C7 herein), SEQ ID NO. 5/SEQ ID NO. 6 (called D3herein), SEQ ID NO. 7/SEQ ID NO. 8 (called E7 herein), SEQ ID NO. 9/SEQID NO. 10 (called F7 herein), SEQ ID NO. 11/SEQ ID NO. 12 (called G3herein), SEQ ID NO. 13/SEQ ID NO. 14 (called G4 herein), SEQ ID NO.15/SEQ ID NO. 16 (called H1 herein), and combinations thereof.

The present disclosure provides a Fab fully human antibody fragment,having a variable domain region from a heavy chain and a variable domainregion from a light chain, wherein the heavy chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 3, SEQ IDNO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 9, SEQ IDNO. 11, SEQ ID NO. 13, SEQ ID NO. 15, and combinations thereof, and thathas a light chain variable domain sequence that is at least 95%identical to the amino acid sequence consisting of SEQ ID NO. 2, SEQ IDNO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ IDNO. 14, SEQ ID NO. 16, and combinations thereof. Preferably, the fullyhuman antibody Fab fragment has both a heavy chain variable domainregion and a light chain variable domain region wherein the antibody hasa heavy chain/light chain variable domain sequence selected from thegroup consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO.4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO.9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO.14, SEQ ID NO. 15/SEQ ID NO. 16, and combinations thereof.

The present disclosure provides a single chain human antibody, having avariable domain region from a heavy chain and a variable domain regionfrom a light chain and a peptide linker connection the heavy chain andlight chain variable domain regions, wherein the heavy chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 9,SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, and combinations thereof,and that has a light chain variable domain sequence that is at least 95%identical to the amino acid sequence consisting of SEQ ID NO. 2, SEQ IDNO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQ IDNO. 14, SEQ ID NO. 16, and combinations thereof. Preferably, the fullyhuman single chain antibody has both a heavy chain variable domainregion and a light chain variable domain region, wherein the singlechain fully human antibody has a heavy chain/light chain variable domainsequence selected from the group consisting of SEQ ID NO. 1/SEQ ID NO.2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO.7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12,SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, andcombinations thereof.

The present disclosure further provides a method for treating orpreventing the S. aureus infection comprising administering an anti-AIP2polypeptide, wherein the anti-AIP2 polypeptide is selected from thegroup consisting of a fully human antibody of an IgG class that binds toa AIP2 epitope with a binding affinity of at least 10⁻⁶M, a Fab fullyhuman antibody fragment, having a variable domain region from a heavychain and a variable domain region from a light chain, a single chainhuman antibody, having a variable domain region from a heavy chain and avariable domain region from a light chain and a peptide linkerconnection the heavy chain and light chain variable domain regions, andcombinations thereof;

wherein the fully human antibody has a heavy chain variable domainsequence that is at least 95% identical to the amino acid sequencesselected from the group consisting of and combinations thereof, and thathas a light chain variable domain sequence that is at least 95%identical to the amino acid sequences selected from the group consistingof SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO.10, SEQ ID NO. 12, SEQ ID NO. 14, SEQ ID NO. 16, and combinationsthereof;

wherein the Fab fully human antibody fragment has the heavy chainvariable domain sequence that is at least 95% identical to the aminoacid sequences selected from the group consisting of SEQ ID NO. 1, SEQID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ IDNO. 9, SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, and combinationsthereof, and that has the light chain variable domain sequence that isat least 95% identical to the amino acid sequence consisting SEQ ID NO.2, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO.12, SEQ ID NO. 14, SEQ ID NO. 16, and combinations thereof; and

wherein the single chain human antibody has the heavy chain variabledomain sequence that is at least 95% identical to the amino acidsequences selected from the group consisting of SEQ ID NO. 1, SEQ ID NO.3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 9,SEQ ID NO. 11, SEQ ID NO. 13, SEQ ID NO. 15, and combinations thereof,and that has the light chain variable domain sequence that is at least95% identical to the amino acid sequence consisting of SEQ ID NO. 2, SEQID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, SEQID NO. 14, SEQ ID NO. 16, and combinations thereof.

Preferably, the fully human antibody has both a heavy chain and a lightchain wherein the antibody has a heavy chain/light chain variable domainsequence selected from the group consisting of SEQ ID NO. 1/SEQ ID NO.2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO.7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12,SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO. 16, andcombinations thereof. Preferably, the fully human antibody Fab fragmenthas both a heavy chain variable domain region and a light chain variabledomain region wherein the antibody has a heavy chain/light chainvariable domain sequence selected from the group consisting of SEQ IDNO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO. 4, SEQ ID NO. 5/SEQ ID NO.6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO. 9/SEQ ID NO. 10, SEQ ID NO.11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO. 14, SEQ ID NO. 15/SEQ ID NO.16, and combinations thereof. Preferably, the fully human single chainantibody has both a heavy chain variable domain region and a light chainvariable domain region, wherein the single chain fully human antibodyhas a heavy chain/light chain variable domain sequence selected from thegroup consisting of SEQ ID NO. 1/SEQ ID NO. 2, SEQ ID NO. 3/SEQ ID NO.4, SEQ ID NO. 5/SEQ ID NO. 6, SEQ ID NO. 7/SEQ ID NO. 8, SEQ ID NO.9/SEQ ID NO. 10, SEQ ID NO. 11/SEQ ID NO. 12, SEQ ID NO. 13/SEQ ID NO.14, SEQ ID NO. 15/SEQ ID NO. 16, and combinations thereof.

Preferably, method for treating or preventing the S. aureus infection iscaused by a MRSA bacteria.

An “antigen binding protein” is a protein comprising a portion thatbinds to an antigen and, optionally, a scaffold or framework portionthat allows the antigen binding portion to adopt a conformation thatpromotes binding of the antigen binding protein to the antigen. Examplesof antigen binding proteins include antibodies, antibody fragments(e.g., an antigen binding portion of an antibody), antibody derivatives,and antibody analogs. The antigen binding protein can comprise, forexample, an alternative protein scaffold or artificial scaffold withgrafted CDRs or CDR derivatives. Such scaffolds include, but are notlimited to, antibody-derived scaffolds comprising mutations introducedto, for example, stabilize the three-dimensional structure of theantigen binding protein as well as wholly synthetic scaffoldscomprising, for example, a biocompatible polymer. See, for example,Korndorfer et al., 2003, Proteins: Structure, Function, andBioinformatics, Volume 53, Issue 1:121-129; Roque et al., 2004,Biotechnol. Prog. 20:639-654. In addition, peptide antibody mimetics(“PAMs”) can be used, as well as scaffolds based on antibody mimeticsutilizing fibronection components as a scaffold.

An antigen binding protein can have, for example, the structure of anaturally occurring immunoglobulin. An “immunoglobulin” is a tetramericmolecule. In a naturally occurring immunoglobulin, each tetramer iscomposed of two identical pairs of polypeptide chains, each pair havingone “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function. Human lightchains are classified as kappa or lambda light chains. Heavy chains areclassified as mu, delta, gamma, alpha, or epsilon, and define theantibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 more amino acids. See generally,Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, N.Y.(1989)) (incorporated by reference in its entirety for all purposes).The variable regions of each light/heavy chain pair form the antibodybinding site such that an intact immunoglobulin has two binding sites.

The variable regions of naturally occurring immunoglobulin chainsexhibit the same general structure of relatively conserved frameworkregions (FR) joined by three hypervariable regions, also calledcomplementarity determining regions or CDRs. From N-terminus toC-terminus, both light and heavy chains comprise the domains FR1, CDR1,FR2, CDR2, FR3, CDR3 and FR4. The assignment of amino acids to eachdomain is in accordance with the definitions of Kabat et al. inSequences of Proteins of Immunological Interest, 5^(th) Ed., US Dept. ofHealth and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991.Other numbering systems for the amino acids in immunoglobulin chainsinclude IMGT® (international ImMunoGeneTics information system; Lefrancet al, Dev. Comp. Immunol. 29:185-203; 2005) and AHo (Honegger andPluckthun, J. Mol. Biol. 309(3):657-670; 2001).

Antibodies can be obtained from sources such as serum or plasma thatcontain immunoglobulins having varied antigenic specificity. If suchantibodies are subjected to affinity purification, they can be enrichedfor a particular antigenic specificity. Such enriched preparations ofantibodies usually are made of less than about 10% antibody havingspecific binding activity for the particular antigen. Subjecting thesepreparations to several rounds of affinity purification can increase theproportion of antibody having specific binding activity for the antigen.Antibodies prepared in this manner are often referred to as“monospecific.” Monospecfic antibody preparations can be made up ofabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,99%, or 99.9% antibody having specific binding activity for theparticular antigen.

An “antibody” refers to an intact immunoglobulin or to an antigenbinding portion thereof that competes with the intact antibody forspecific binding, unless otherwise specified. Antigen binding portionsmay be produced by recombinant DNA techniques or by enzymatic orchemical cleavage of intact antibodies. Antigen binding portionsinclude, inter alia, Fab, Fab′, F(ab′)₂, Fv, domain antibodies (dAbs),and complementarity determining region (CDR) fragments, single-chainantibodies (scFv), chimeric antibodies, diabodies, triabodies,tetrabodies, and polypeptides that contain at least a portion of animmunoglobulin that is sufficient to confer specific antigen binding tothe polypeptide.

A Fab fragment is a monovalent fragment having the V_(L), V_(H), C_(L)and C_(H1) domains; a F(ab′)₂ fragment is a bivalent fragment having twoFab fragments linked by a disulfide bridge at the hinge region; a Fdfragment has the V_(H) and C_(H1) domains; an Fv fragment has the V_(L)and V_(H) domains of a single arm of an antibody; and a dAb fragment hasa V_(H) domain, a V_(L) domain, or an antigen-binding fragment of aV_(H) or VL domain (U.S. Pat. Nos. 6,846,634; 6,696,245, US App. Pub.20/0202512; 2004/0202995; 2004/0038291; 2004/0009507; 2003/0039958, andWard et al., Nature 341:544-546, 1989).

A single-chain antibody (scFv) is an antibody in which a V_(L) and aV_(H) region are joined via a linker (e.g., a synthetic sequence ofamino acid residues) to form a continuous protein chain wherein thelinker is long enough to allow the protein chain to fold back on itselfand form a monovalent antigen binding site (see, e.g., Bird et al.,1988, Science 242:423-26 and Huston et al., 1988, Proc. Natl. Acad. Sci.USA 85:5879-83). Diabodies are bivalent antibodies comprising twopolypeptide chains, wherein each polypeptide chain comprises V_(H) andV_(L) domains joined by a linker that is too short to allow for pairingbetween two domains on the same chain, thus allowing each domain to pairwith a complementary domain on another polypeptide chain (see, e.g.,Holliger et al., 1993, Proc. Natl. Acad. Sci. USA 90:6444-48, and Poljaket al., 1994, Structure 2:1121-23). If the two polypeptide chains of adiabody are identical, then a diabody resulting from their pairing willhave two identical antigen binding sites. Polypeptide chains havingdifferent sequences can be used to make a diabody with two differentantigen binding sites. Similarly, tribodies and tetrabodies areantibodies comprising three and four polypeptide chains, respectively,and forming three and four antigen binding sites, respectively, whichcan be the same or different.

Complementarity determining regions (CDRs) and framework regions (FR) ofa given antibody may be identified using the system described by Kabatet al. supra; Lefranc et al., supra and/or Honegger and Pluckthun,supra. One or more CDRs may be incorporated into a molecule eithercovalently or noncovalently to make it an antigen binding protein. Anantigen binding protein may incorporate the CDR(s) as part of a largerpolypeptide chain, may covalently link the CDR(s) to another polypeptidechain, or may incorporate the CDR(s) noncovalently. The CDRs permit theantigen binding protein to specifically bind to a particular antigen ofinterest.

An antigen binding protein may have one or more binding sites. If thereis more than one binding site, the binding sites may be identical to oneanother or may be different. For example, a naturally occurring humanimmunoglobulin typically has two identical binding sites, while a“bispecific” or “bifunctional” antibody has two different binding sites.

The term “human antibody” includes all antibodies that have one or morevariable and constant regions derived from human immunoglobulinsequences. In one embodiment, all of the variable and constant domainsare derived from human immunoglobulin sequences (a fully humanantibody). These antibodies may be prepared in a variety of ways,examples of which are described below, including through theimmunization with an antigen of interest of a mouse that is geneticallymodified to express antibodies derived from human heavy and/or lightchain-encoding genes.

A humanized antibody has a sequence that differs from the sequence of anantibody derived from a non-human species by one or more amino acidsubstitutions, deletions, and/or additions, such that the humanizedantibody is less likely to induce an immune response, and/or induces aless severe immune response, as compared to the non-human speciesantibody, when it is administered to a human subject. In one embodiment,certain amino acids in the framework and constant domains of the heavyand/or light chains of the non-human species antibody are mutated toproduce the humanized antibody. In another embodiment, the constantdomain(s) from a human antibody are fused to the variable domain(s) of anon-human species. In another embodiment, one or more amino acidresidues in one or more CDR sequences of a non-human antibody arechanged to reduce the likely immunogenicity of the non-human antibodywhen it is administered to a human subject, wherein the changed aminoacid residues either are not critical for immunospecific binding of theantibody to its antigen, or the changes to the amino acid sequence thatare made are conservative changes, such that the binding of thehumanized antibody to the antigen is not significantly worse than thebinding of the non-human antibody to the antigen. Examples of how tomake humanized antibodies may be found in U.S. Pat. Nos. 6,054,297,5,886,152 and 5,877,293.

The term “chimeric antibody” refers to an antibody that contains one ormore regions from one antibody and one or more regions from one or moreother antibodies. In one embodiment, one or more of the CDRs are derivedfrom a human anti-AIP2 antibody. In another embodiment, all of the CDRsare derived from a human anti-AIP2 antibody. In another embodiment, theCDRs from more than one human anti-AIP2 antibodies are mixed and matchedin a chimeric antibody. For instance, a chimeric antibody may comprise aCDR1 from the light chain of a first human anti-PAR-2 antibody, a CDR2and a CDR3 from the light chain of a second human anti-AIP2 antibody,and the CDRs from the heavy chain from a third anti-AIP2 antibody. Othercombinations are possible.

Further, the framework regions may be derived from one of the sameanti-AIP2 antibodies, from one or more different antibodies, such as ahuman antibody, or from a humanized antibody. In one example of achimeric antibody, a portion of the heavy and/or light chain isidentical with, homologous to, or derived from an antibody from aparticular species or belonging to a particular antibody class orsubclass, while the remainder of the chain(s) is/are identical with,homologous to, or derived from an antibody (-ies) from another speciesor belonging to another antibody class or subclass. Also included arefragments of such antibodies that exhibit the desired biologicalactivity (i.e., the ability to specifically bind AIP2).

A “neutralizing antibody” or an “inhibitory antibody” is an antibodythat inhibits the proteolytic activation of AIP2 when an excess of theanti-AIP2 antibody reduces the amount of activation by at least about20% using an assay such as those described herein in the Examples. Invarious embodiments, the antigen binding protein reduces the amount ofamount of proteolytic activation of AIP2 by at least 30%, 40%, 50%, 60%,70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, and 99.9%.

Fragments or analogs of antibodies can be readily prepared by those ofordinary skill in the art following the teachings of this specificationand using techniques known in the art. Preferred amino- andcarboxy-termini of fragments or analogs occur near boundaries offunctional domains. Structural and functional domains can be identifiedby comparison of the nucleotide and/or amino acid sequence data topublic or proprietary sequence databases. Computerized comparisonmethods can be used to identify sequence motifs or predicted proteinconformation domains that occur in other proteins of known structureand/or function. Methods to identify protein sequences that fold into aknown three-dimensional structure are known. See, Bowie et al., 1991,Science 253:164.

A “CDR grafted antibody” is an antibody comprising one or more CDRsderived from an antibody of a particular species or isotype and theframework of another antibody of the same or different species orisotype.

A “multi-specific antibody” is an antibody that recognizes more than oneepitope on one or more antigens. A subclass of this type of antibody isa “bi-specific antibody” which recognizes two distinct epitopes on thesame or different antigens.

An antigen binding protein “specifically binds” to an antigen (e.g.,human AIP2) if it binds to the antigen with a dissociation constant of 1nanomolar or less.

An “antigen binding domain,” “antigen binding region,” or “antigenbinding site” is a portion of an antigen binding protein that containsamino acid residues (or other moieties) that interact with an antigenand contribute to the antigen binding protein's specificity and affinityfor the antigen. For an antibody that specifically binds to its antigen,this will include at least part of at least one of its CDR domains.

An “epitope” is the portion of a molecule that is bound by an antigenbinding protein (e.g., by an antibody). An epitope can comprisenon-contiguous portions of the molecule (e.g., in a polypeptide, aminoacid residues that are not contiguous in the polypeptide's primarysequence but that, in the context of the polypeptide's tertiary andquaternary structure, are near enough to each other to be bound by anantigen binding protein).

The “percent identity” of two polynucleotide or two polypeptidesequences is determined by comparing the sequences using the GAPcomputer program (a part of the GCG Wisconsin Package, version 10.3(Accelrys, San Diego, Calif.)) using its default parameters.

The terms “polynucleotide,” “oligonucleotide” and “nucleic acid” areused interchangeably throughout and include DNA molecules (e.g., cDNA orgenomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNAgenerated using nucleotide analogs (e.g., peptide nucleic acids andnon-naturally occurring nucleotide analogs), and hybrids thereof. Thenucleic acid molecule can be single-stranded or double-stranded. In oneembodiment, the nucleic acid molecules of the invention comprise acontiguous open reading frame encoding an antibody, or a fragment,derivative, mutein, or variant thereof.

Two single-stranded polynucleotides are “the complement” of each otherif their sequences can be aligned in an anti-parallel orientation suchthat every nucleotide in one polynucleotide is opposite itscomplementary nucleotide in the other polynucleotide, without theintroduction of gaps, and without unpaired nucleotides at the 5′ or the3′ end of either sequence. A polynucleotide is “complementary” toanother polynucleotide if the two polynucleotides can hybridize to oneanother under moderately stringent conditions. Thus, a polynucleotidecan be complementary to another polynucleotide without being itscomplement.

A “vector” is a nucleic acid that can be used to introduce anothernucleic acid linked to it into a cell. One type of vector is a“plasmid,” which refers to a linear or circular double stranded DNAmolecule into which additional nucleic acid segments can be ligated.Another type of vector is a viral vector (e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses), whereinadditional DNA segments can be introduced into the viral genome. Certainvectors are capable of autonomous replication in a host cell into whichthey are introduced (e.g., bacterial vectors comprising a bacterialorigin of replication and episomal mammalian vectors). Other vectors(e.g., non-episomal mammalian vectors) are integrated into the genome ofa host cell upon introduction into the host cell, and thereby arereplicated along with the host genome. An “expression vector” is a typeof vector that can direct the expression of a chosen polynucleotide.

A nucleotide sequence is “operably linked” to a regulatory sequence ifthe regulatory sequence affects the expression (e.g., the level, timing,or location of expression) of the nucleotide sequence. A “regulatorysequence” is a nucleic acid that affects the expression (e.g., thelevel, timing, or location of expression) of a nucleic acid to which itis operably linked. The regulatory sequence can, for example, exert itseffects directly on the regulated nucleic acid, or through the action ofone or more other molecules (e.g., polypeptides that bind to theregulatory sequence and/or the nucleic acid). Examples of regulatorysequences include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Further examples of regulatorysequences are described in, for example, Goeddel, 1990, Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.and Baron et al., 1995, Nucleic Acids Res. 23:3605-06.

A “host cell” is a cell that can be used to express a nucleic acid,e.g., a nucleic acid of the invention. A host cell can be a prokaryote,for example, E. coli, or it can be a eukaryote, for example, asingle-celled eukaryote (e.g., a yeast or other fungus), a plant cell(e.g., a tobacco or tomato plant cell), an animal cell (e.g., a humancell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or aninsect cell) or a hybridoma. Examples of host cells include the COS-7line of monkey kidney cells (ATCC CRL 1651) (see Gluzman et al., 1981,Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinesehamster ovary (CHO) cells or their derivatives such as Veggie CHO andrelated cell lines which grow in serum-free media (see Rasmussen et al.,1998, Cytotechnology 28:31) or CHO strain DX-B11, which is deficient inDHFR (see Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20),HeLa cells, BHK (ATCC CRL 10) cell lines, the CV1/EBNA cell line derivedfrom the African green monkey kidney cell line CV1 (ATCC CCL 70) (seeMcMahan et al., 1991, EMBO J. 10:2821), human embryonic kidney cellssuch as 293,293 EBNA or MSR 293, human epidermal A431 cells, humanColo205 cells, other transformed primate cell lines, normal diploidcells, cell strains derived from in vitro culture of primary tissue,primary explants, HL-60, U937, HaK or Jurkat cells. Typically, a hostcell is a cultured cell that can be transformed or transfected with apolypeptide-encoding nucleic acid, which can then be expressed in thehost cell. The phrase “recombinant host cell” can be used to denote ahost cell that has been transformed or transfected with a nucleic acidto be expressed. A host cell also can be a cell that comprises thenucleic acid but does not express it at a desired level unless aregulatory sequence is introduced into the host cell such that itbecomes operably linked with the nucleic acid. It is understood that theterm host cell refers not only to the particular subject cell but alsoto the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to, e.g., mutationor environmental influence, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

Polypeptides of the present disclosure can be produced using anystandard methods known in the art. In one example, the polypeptides areproduced by recombinant DNA methods by inserting a nucleic acid sequence(e.g., a cDNA) encoding the polypeptide into a recombinant expressionvector and expressing the DNA sequence under conditions promotingexpression.

Nucleic acids encoding any of the various polypeptides disclosed hereinmay be synthesized chemically. Codon usage may be selected so as toimprove expression in a cell. Such codon usage will depend on the celltype selected. Specialized codon usage patterns have been developed forE. coli and other bacteria, as well as mammalian cells, plant cells,yeast cells and insect cells. See for example: Mayfield et al., Proc.Natl. Acad. Sci. USA. 2003 100(2):438-42; Sinclair et al. Protein Expr.Purif. 2002 (1):96-105; Connell N D. Curr. Opin. Biotechnol. 200112(5):446-9; Makrides et al. Microbiol. Rev. 1996 60(3):512-38; andSharp et al. Yeast. 1991 7(7):657-78.

General techniques for nucleic acid manipulation are described forexample in Sambrook et al., Molecular Cloning: A Laboratory Manual,Vols. 1-3, Cold Spring Harbor Laboratory Press, 2 ed., 1989, or F.Ausubel et al., Current Protocols in Molecular Biology (Green Publishingand Wiley-Interscience: New York, 1987) and periodic updates, hereinincorporated by reference. The DNA encoding the polypeptide is operablylinked to suitable transcriptional or translational regulatory elementsderived from mammalian, viral, or insect genes. Such regulatory elementsinclude a transcriptional promoter, an optional operator sequence tocontrol transcription, a sequence encoding suitable mRNA ribosomalbinding sites, and sequences that control the termination oftranscription and translation. The ability to replicate in a host,usually conferred by an origin of replication, and a selection gene tofacilitate recognition of transformants is additionally incorporated.

The recombinant DNA can also include any type of protein tag sequencethat may be useful for purifying the protein. Examples of protein tagsinclude but are not limited to a histidine tag, a FLAG tag, a myc tag,an HA tag, or a GST tag. Appropriate cloning and expression vectors foruse with bacterial, fungal, yeast, and mammalian cellular hosts can befound in Cloning Vectors: A Laboratory Manual, (Elsevier, N.Y., 1985).

The expression construct is introduced into the host cell using a methodappropriate to the host cell. A variety of methods for introducingnucleic acids into host cells are known in the art, including, but notlimited to, electroporation; transfection employing calcium chloride,rubidium chloride, calcium phosphate, DEAE-dextran, or other substances;microprojectile bombardment; lipofection; and infection (where thevector is an infectious agent). Suitable host cells include prokaryotes,yeast, mammalian cells, or bacterial cells.

Suitable bacteria include gram negative or gram positive organisms, forexample, E. coli or Bacillus spp. Yeast, preferably from theSaccharomyces species, such as S. cerevisiae, may also be used forproduction of polypeptides. Various mammalian or insect cell culturesystems can also be employed to express recombinant proteins.Baculovirus systems for production of heterologous proteins in insectcells are reviewed by Luckow and Summers, (Bio/Technology, 6:47, 1988).Examples of suitable mammalian host cell lines include endothelialcells, COS-7 monkey kidney cells, CV-1, L cells, C127, 3T3, Chinesehamster ovary (CHO), human embryonic kidney cells, HeLa, 293, 293T, andBHK cell lines. Purified polypeptides are prepared by culturing suitablehost/vector systems to express the recombinant proteins. For manyapplications, the small size of many of the polypeptides disclosedherein would make expression in E. coli as the preferred method forexpression. The protein is then purified from culture media or cellextracts.

Proteins disclosed herein can also be produced using cell-translationsystems. For such purposes the nucleic acids encoding the polypeptidemust be modified to allow in vitro transcription to produce mRNA and toallow cell-free translation of the mRNA in the particular cell-freesystem being utilized (eukaryotic such as a mammalian or yeast cell-freetranslation system or prokaryotic such as a bacterial cell-freetranslation system.

AIP2-binding polypeptides can also be produced by chemical synthesis(e.g., by the methods described in Solid Phase Peptide Synthesis, 2nded., 1984, The Pierce Chemical Co., Rockford, Ill.). Modifications tothe protein can also be produced by chemical synthesis.

The polypeptides of the present disclosure can be purified byisolation/purification methods for proteins generally known in the fieldof protein chemistry. Non-limiting examples include extraction,recrystallization, salting out (e.g., with ammonium sulfate or sodiumsulfate), centrifugation, dialysis, ultrafiltration, adsorptionchromatography, ion exchange chromatography, hydrophobic chromatography,normal phase chromatography, reversed-phase chromatography, gelfiltration, gel permeation chromatography, affinity chromatography,electrophoresis, countercurrent distribution or any combinations ofthese. After purification, polypeptides may be exchanged into differentbuffers and/or concentrated by any of a variety of methods known to theart, including, but not limited to, filtration and dialysis.

The purified polypeptide is preferably at least 85% pure, morepreferably at least 95% pure, and most preferably at least 98% pure.Regardless of the exact numerical value of the purity, the polypeptideis sufficiently pure for use as a pharmaceutical product.

Post-Translational Modifications of Polypeptides

In certain embodiments, the binding polypeptides of the invention mayfurther comprise post-translational modifications. Exemplarypost-translational protein modifications include phosphorylation,acetylation, methylation, ADP-ribosylation, ubiquitination,glycosylation, carbonylation, sumoylation, biotinylation or addition ofa polypeptide side chain or of a hydrophobic group. As a result, themodified soluble polypeptides may contain non-amino acid elements, suchas lipids, poly- or mono-saccharide, and phosphates. A preferred form ofglycosylation is sialylation, which conjugates one or more sialic acidmoieties to the polypeptide. Sialic acid moieties improve solubility andserum half-life while also reducing the possible immunogeneticity of theprotein. See Raju et al. Biochemistry. 2001 31; 40(30):8868-76.

In one embodiment, modified forms of the subject soluble polypeptidescomprise linking the subject soluble polypeptides to nonproteinaceouspolymers. In one embodiment, the polymer is polyethylene glycol (“PEG”),polypropylene glycol, or polyoxyalkylenes, in the manner as set forth inU.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or4,179,337.

PEG is a water soluble polymer that is commercially available or can beprepared by ring-opening polymerization of ethylene glycol according tomethods well known in the art (Sandler and Karo, Polymer Synthesis,Academic Press, New York, Vol. 3, pages 138-161). The term “PEG” is usedbroadly to encompass any polyethylene glycol molecule, without regard tosize or to modification at an end of the PEG, and can be represented bythe formula: X—O(CH₂CH₂O)_(n)—CH₂CH₂OH (1), where n is 20 to 2300 and Xis H or a terminal modification, e.g., a C₁₋₄ alkyl. In one embodiment,the PEG of the invention terminates on one end with hydroxy or methoxy,i.e., X is H or CH₃ (“methoxy PEG”). A PEG can contain further chemicalgroups which are necessary for binding reactions; which results from thechemical synthesis of the molecule; or which is a spacer for optimaldistance of parts of the molecule. In addition, such a PEG can consistof one or more PEG side-chains which are linked together. PEGs with morethan one PEG chain are called multiarmed or branched PEGs. Branched PEGscan be prepared, for example, by the addition of polyethylene oxide tovarious polyols, including glycerol, pentaerythriol, and sorbitol. Forexample, a four-armed branched PEG can be prepared from pentaerythrioland ethylene oxide. Branched PEG are described in, for example, EP-A 0473 084 and U.S. Pat. No. 5,932,462. One form of PEGs includes two PEGside-chains (PEG2) linked via the primary amino groups of a lysine(Monfardini et al., Bioconjugate Chem. 6 (1995) 62-69).

The serum clearance rate of PEG-modified polypeptide may be decreased byabout 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or even 90%, relative tothe clearance rate of the unmodified binding polypeptide. ThePEG-modified polypeptide may have a half-life (t_(1/2)) which isenhanced relative to the half-life of the unmodified protein. Thehalf-life of PEG-binding polypeptide may be enhanced by at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%,250%, 300%, 400% or 500%, or even by 1000% relative to the half-life ofthe unmodified binding polypeptide. In some embodiments, the proteinhalf-life is determined in vitro, such as in a buffered saline solutionor in serum. In other embodiments, the protein half-life is an in vivohalf life, such as the half-life of the protein in the serum or otherbodily fluid of an animal.

Therapeutic Formulations and Modes of Administration

The present disclosure features method for treating or preventing the S.aureus infection comprising administering an anti-AIP2 polypeptide.Techniques and dosages for administration vary depending on the type ofspecific polypeptide and the specific condition being treated but can bereadily determined by the skilled artisan. In general, regulatoryagencies require that a protein reagent to be used as a therapeutic isformulated so as to have acceptably low levels of pyrogens. Accordingly,therapeutic formulations will generally be distinguished from otherformulations in that they are substantially pyrogen free, or at leastcontain no more than acceptable levels of pyrogen as determined by theappropriate regulatory agency (e.g., FDA).

Therapeutic compositions of the present disclosure may be administeredwith a pharmaceutically acceptable diluent, carrier, or excipient, inunit dosage form. Administration may be parenteral (e.g., intravenous,subcutaneous), oral, or topical, as non-limiting examples. In addition,any gene therapy technique, using nucleic acids encoding thepolypeptides of the invention, may be employed, such as naked DNAdelivery, recombinant genes and vectors, cell-based delivery, includingex vivo manipulation of patients' cells, and the like.

The composition can be in the form of a pill, tablet, capsule, liquid,or sustained release tablet for oral administration; or a liquid forintravenous, subcutaneous or parenteral administration; gel, lotion,ointment, cream, or a polymer or other sustained release vehicle forlocal administration.

Methods well known in the art for making formulations are found, forexample, in “Remington: The Science and Practice of Pharmacy” (20th ed.,ed. A. R. Gennaro A R., 2000, Lippincott Williams & Wilkins,Philadelphia, Pa.). Formulations for parenteral administration may, forexample, contain excipients, sterile water, saline, polyalkylene glycolssuch as polyethylene glycol, oils of vegetable origin, or hydrogenatednapthalenes. Biocompatible, biodegradable lactide polymer,lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylenecopolymers may be used to control the release of the compounds.Nanoparticulate formulations (e.g., biodegradable nanoparticles, solidlipid nanoparticles, liposomes) may be used to control thebiodistribution of the compounds. Other potentially useful parenteraldelivery systems include ethylene-vinyl acetate copolymer particles,osmotic pumps, implantable infusion systems, and liposomes. Theconcentration of the compound in the formulation varies depending upon anumber of factors, including the dosage of the drug to be administered,and the route of administration.

The polypeptide may be optionally administered as a pharmaceuticallyacceptable salt, such as non-toxic acid addition salts or metalcomplexes that are commonly used in the pharmaceutical industry.Examples of acid addition salts include organic acids such as acetic,lactic, pamoic, maleic, citric, malic, ascorbic, succinic, benzoic,palmitic, suberic, salicylic, tartaric, methanesulfonic,toluenesulfonic, or trifluoroacetic acids or the like; polymeric acidssuch as tannic acid, carboxymethyl cellulose, or the like; and inorganicacid such as hydrochloric acid, hydrobromic acid, sulfuric acidphosphoric acid, or the like. Metal complexes include zinc, iron, andthe like. In one example, the polypeptide is formulated in the presenceof sodium acetate to increase thermal stability.

Formulations for oral use include tablets containing the activeingredient(s) in a mixture with non-toxic pharmaceutically acceptableexcipients. These excipients may be, for example, inert diluents orfillers (e.g., sucrose and sorbitol), lubricating agents, glidants, andanti-adhesives (e.g., magnesium stearate, zinc stearate, stearic acid,silicas, hydrogenated vegetable oils, or talc).

Formulations for oral use may also be provided as chewable tablets, oras hard gelatin capsules wherein the active ingredient is mixed with aninert solid diluent, or as soft gelatin capsules wherein the activeingredient is mixed with water or an oil medium.

A therapeutically effective dose refers to a dose that produces thetherapeutic effects for which it is administered. The exact dose willdepend on the disorder to be treated, and may be ascertained by oneskilled in the art using known techniques. In general, the polypeptideis administered at about 0.01 μg/kg to about 50 mg/kg per day,preferably 0.01 mg/kg to about 30 mg/kg per day, most preferably 0.1mg/kg to about 20 mg/kg per day. The polypeptide may be given daily(e.g., once, twice, three times, or four times daily) or preferably lessfrequently (e.g., weekly, every two weeks, every three weeks, monthly,or quarterly). In addition, as is known in the art, adjustments for ageas well as the body weight, general health, sex, diet, time ofadministration, drug interaction, and the severity of the disease may benecessary, and will be ascertainable with routine experimentation bythose skilled in the art.

Exemplary Uses

An AIP2 binding polypeptide can be administered alone or in combinationwith one or more additional therapies such as chemotherapy radiotherapy,immunotherapy, surgical intervention, or any combination of these.Long-term therapy is equally possible as is adjuvant therapy in thecontext of other treatment strategies, as described above.

In certain embodiments of such methods, one or more polypeptidetherapeutic agents can be administered, together (simultaneously) or atdifferent times (sequentially). In addition, polypeptide therapeuticagents can be administered with another type of compounds for treatingcancer or for inhibiting angiogenesis.

In certain embodiments, the subject anti-AIP2 antibodies agents of theinvention can be used alone.

In certain embodiments, the binding polypeptides of fragments thereofcan be labeled or unlabeled for diagnostic purposes. Typically,diagnostic assays entail detecting the formation of a complex resultingfrom the binding of a binding polypeptide to AIP2. The bindingpolypeptides or fragments can be directly labeled, similar toantibodies. A variety of labels can be employed, including, but notlimited to, radionuclides, fluorescers, enzymes, enzyme substrates,enzyme cofactors, enzyme inhibitors and ligands (e.g., biotin, haptens).Numerous appropriate immunoassays are known to the skilled artisan (see,for example, U.S. Pat. Nos. 3,817,827; 3,850,752; 3,901,654; and4,098,876). When unlabeled, the binding polypeptides can be used inassays, such as agglutination assays. Unlabeled binding polypeptides canalso be used in combination with another (one or more) suitable reagentwhich can be used to detect the binding polypeptide, such as a labeledantibody reactive with the binding polypeptide or other suitable reagent(e.g., labeled protein A).

In one embodiment, the binding polypeptides of the present invention canbe utilized in enzyme immunoassays, wherein the subject polypeptides areconjugated to an enzyme. When a biological sample comprising a AIP2protein is combined with the subject binding polypeptides, bindingoccurs between the binding polypeptides and the AIP2 protein. In oneembodiment, a sample containing cells expressing an AIP2 protein (e.g.,endothelial cells) is combined with the subject antibodies, and bindingoccurs between the binding polypeptides and cells bearing a AIP2 proteinrecognized by the binding polypeptide. These bound cells can beseparated from unbound reagents and the presence of the bindingpolypeptide-enzyme conjugate specifically bound to the cells can bedetermined, for example, by contacting the sample with a substrate ofthe enzyme which produces a color or other detectable change when actedon by the enzyme. In another embodiment, the subject bindingpolypeptides can be unlabeled, and a second, labeled polypeptide (e.g.,an antibody) can be added which recognizes the subject bindingpolypeptide.

In certain aspects, kits for use in detecting the presence of an AIP2protein in a biological sample can also be prepared. Such kits willinclude an AIP2 binding polypeptide which binds to an AIP2 protein orportion of said receptor, as well as one or more ancillary reagentssuitable for detecting the presence of a complex between the bindingpolypeptide and the receptor protein or portions thereof. Thepolypeptide compositions of the present invention can be provided inlyophilized form, either alone or in combination with additionalantibodies specific for other epitopes. The binding polypeptides and/orantibodies, which can be labeled or unlabeled, can be included in thekits with adjunct ingredients (e.g., buffers, such as Tris, phosphateand carbonate, stabilizers, excipients, biocides and/or inert proteins,e.g., bovine serum albumin). For example, the binding polypeptidesand/or antibodies can be provided as a lyophilized mixture with theadjunct ingredients, or the adjunct ingredients can be separatelyprovided for combination by the user. Generally these adjunct materialswill be present in less than about 5% weight based on the amount ofactive binding polypeptide or antibody, and usually will be present in atotal amount of at least about 0.001% weight based on polypeptide orantibody concentration. Where a second antibody capable of binding tothe binding polypeptide is employed, such antibody can be provided inthe kit, for instance in a separate vial or container. The secondantibody, if present, is typically labeled, and can be formulated in ananalogous manner with the antibody formulations described above.

Polypeptide sequences are indicated using standard one- or three-letterabbreviations. Unless otherwise indicated, each polypeptide sequence hasamino termini at the left and a carboxy termini at the right; eachsingle-stranded nucleic acid sequence, and the top strand of eachdouble-stranded nucleic acid sequence, has a 5′ termini at the left anda 3′ termini at the right. A particular polypeptide sequence also can bedescribed by explaining how it differs from a reference sequence.

The following terms, unless otherwise indicated, shall be understood tohave the following meanings:

The terms “peptide,” “polypeptide” and “protein” each refers to amolecule comprising two or more amino acid residues joined to each otherby peptide bonds. These terms encompass, e.g., native and artificialproteins, protein fragments and polypeptide analogs (such as muteins,variants, and fusion proteins) of a protein sequence as well aspost-translationally, or otherwise covalently or non-covalently,modified proteins. A peptide, polypeptide, or protein may be monomericor polymeric.

A “variant” of a polypeptide (for example, an antibody) comprises anamino acid sequence wherein one or more amino acid residues are insertedinto, deleted from and/or substituted into the amino acid sequencerelative to another polypeptide sequence. Disclosed variants include,for example, fusion proteins.

A “derivative” of a polypeptide is a polypeptide (e.g., an antibody)that has been chemically modified, e.g., via conjugation to anotherchemical moiety (such as, for example, polyethylene glycol or albumin,e.g., human serum albumin), phosphorylation, and glycosylation. Unlessotherwise indicated, the term “antibody” includes, in addition toantibodies comprising two full-length heavy chains and two full-lengthlight chains, derivatives, variants, fragments, and muteins thereof,examples of which are described below.

An “antigen binding protein” is a protein comprising a portion thatbinds to an antigen and, optionally, a scaffold or framework portionthat allows the antigen binding portion to adopt a conformation thatpromotes binding of the antigen binding protein to the antigen. Examplesof antigen binding proteins include antibodies, antibody fragments(e.g., an antigen binding portion of an antibody), antibody derivatives,and antibody analogs. The antigen binding protein can comprise, forexample, an alternative protein scaffold or artificial scaffold withgrafted CDRs or CDR derivatives. Such scaffolds include, but are notlimited to, antibody-derived scaffolds comprising mutations introducedto, for example, stabilize the three-dimensional structure of theantigen binding protein as well as wholly synthetic scaffoldscomprising, for example, a biocompatible polymer. See, for example,Korndorfer et al., 2003, Proteins: Structure, Function, andBioinformatics, Volume 53, Issue 1:121-129; Roque et al., 2004,Biotechnol. Prog. 20:639-654. In addition, peptide antibody mimetics(“PAMs”) can be used, as well as scaffolds based on antibody mimeticsutilizing fibronection components as a scaffold.

An antigen binding protein can have, for example, the structure of anaturally occurring immunoglobulin. An “immunoglobulin” is a tetramericmolecule. In a naturally occurring immunoglobulin, each tetramer iscomposed of two identical pairs of polypeptide chains, each pair havingone “light” (about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function. Human lightchains are classified as kappa or lambda light chains. Heavy chains areclassified as mu, delta, gamma, alpha, or epsilon, and define theantibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively.Preferably, the anti-EGFR antibodies disclosed herein are characterizedby their variable domain region sequences in the heavy V_(H) and lightV_(L) amino acid sequences. The preferred antibody is A6 which is akappa IgG antibody. Within light and heavy chains, the variable andconstant regions are joined by a “J” region of about 12 or more aminoacids, with the heavy chain also including a “D” region of about 10 moreamino acids. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed.,2nd ed. Raven Press, N.Y. (1989)). The variable regions of eachlight/heavy chain pair form the antibody binding site such that anintact immunoglobulin has two binding sites.

A “multi-specific antibody” is an antibody that recognizes more than oneepitope on one or more antigens. A subclass of this type of antibody isa “bi-specific antibody” which recognizes two distinct epitopes on thesame or different antigens.

An antigen binding protein “specifically binds” to an antigen (e.g.,human AIP2) if it binds to the antigen with a dissociation constant of 1nanomolar or less.

An “antigen binding domain, “antigen binding region,” or “antigenbinding site” is a portion of an antigen binding protein that containsamino acid residues (or other moieties) that interact with an antigenand contribute to the antigen binding protein's specificity and affinityfor the antigen. For an antibody that specifically binds to its antigen,this will include at least part of at least one of its CDR domains.

An “epitope” is the portion of a molecule that is bound by an antigenbinding protein (e.g., by an antibody). An epitope can comprisenon-contiguous portions of the molecule (e.g., in a polypeptide, aminoacid residues that are not contiguous in the polypeptide's primarysequence but that, in the context of the polypeptide's tertiary andquaternary structure, are near enough to each other to be bound by anantigen binding protein).

The “percent homology” of two polynucleotide or two polypeptidesequences is determined by comparing the sequences using the GAPcomputer program (a part of the GCG Wisconsin Package, version 10.3(Accelrys, San Diego, Calif.)) using its default parameters.

A “host cell” is a cell that can be used to express a nucleic acid. Ahost cell can be a prokaryote, for example, E. coli, or it can be aeukaryote, for example, a single-celled eukaryote (e.g., a yeast orother fungus), a plant cell (e.g., a tobacco or tomato plant cell), ananimal cell (e.g., a human cell, a monkey cell, a hamster cell, a ratcell, a mouse cell, or an insect cell) or a hybridoma. Examples of hostcells include the COS-7 line of monkey kidney cells (ATCC CRL 1651)(Gluzman et al., 1981, Cell 23:175), L cells, C127 cells, 3T3 cells(ATCC CCL 163), Chinese hamster ovary (CHO) cells or their derivativessuch as Veggie CHO and related cell lines which grow in serum-free media(Rasmussen et al., 1998, Cytotechnology 28:31) or CHO strain DX-B11,which is deficient in DHFR (Urlaub et al., 1980, Proc. Natl. Acad. Sci.USA 77:4216-20), HeLa cells, BHK (ATCC CRL 10) cell lines, the CV1/EBNAcell line derived from the African green monkey kidney cell line CV1(ATCC CCL 70) (McMahan et al., 1991, EMBO J. 10:2821), human embryonickidney cells such as 293,293 EBNA or MSR 293, human epidermal A431cells, human Colo205 cells, other transformed primate cell lines, normaldiploid cells, cell strains derived from in vitro culture of primarytissue, primary explants, HL-60, U937, HaK or Jurkat cells. Typically, ahost cell is a cultured cell that can be transformed or transfected witha polypeptide-encoding nucleic acid, which can then be expressed in thehost cell. The phrase “recombinant host cell” can be used to denote ahost cell that has been transformed or transfected with a nucleic acidto be expressed. A host cell also can be a cell that comprises thenucleic acid but does not express it at a desired level unless aregulatory sequence is introduced into the host cell such that itbecomes operably linked with the nucleic acid. It is understood that theterm host cell refers not only to the particular subject cell but alsoto the progeny or potential progeny of such a cell. Because certainmodifications may occur in succeeding generations due to, e.g., mutationor environmental influence, such progeny may not, in fact, be identicalto the parent cell, but are still included within the scope of the termas used herein.

Antigen Binding Proteins

Antigen binding proteins (e.g., antibodies, antibody fragments, antibodyderivatives, antibody muteins, and antibody variants) are polypeptidesthat bind to AIP2.

Oligomers that contain one or more antigen binding proteins may beemployed as AIP2 antagonists. Oligomers may be in the form ofcovalently-linked or non-covalently-linked dimers, trimers, or higheroligomers. Oligomers comprising two or more antigen binding protein arecontemplated for use, with one example being a homodimer. Otheroligomers include heterodimers, homotrimers, heterotrimers,homotetramers, heterotetramers, etc.

One embodiment is directed to oligomers comprising multiple antigenbinding proteins joined via covalent or non-covalent interactionsbetween peptide moieties fused to the antigen binding proteins. Suchpeptides may be peptide linkers (spacers), or peptides that have theproperty of promoting oligomerization. Leucine zippers and certainpolypeptides derived from antibodies are among the peptides that canpromote oligomerization of antigen binding proteins attached thereto, asdescribed in more detail below.

In particular embodiments, the oligomers comprise from two to fourantigen binding proteins. The antigen binding proteins of the oligomermay be in any form, such as any of the forms described above, e.g.,variants or fragments. Preferably, the oligomers comprise antigenbinding proteins that have AIP2 binding activity.

In one embodiment, an oligomer is prepared using polypeptides derivedfrom immunoglobulins. Preparation of Fusion Proteins Comprising CertainHeterologous Polypeptides Fused to Various Portions of antibody-derivedpolypeptides (including the Fc domain) has been described, e.g., byAshkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88:10535; Byrn etal., 1990, Nature 344:677; and Hollenbaugh et al., 1992 “Construction ofImmunoglobulin Fusion Proteins”, in Current Protocols in Immunology,Suppl. 4, pages 10.19.1-10.19.11.

One embodiment is directed to a dimer comprising two fusion proteinscreated by fusing a AIP2 binding fragment of an anti-AIP2 antibody tothe Fc region of an antibody. The dimer can be made by, for example,inserting a gene fusion encoding the fusion protein into an appropriateexpression vector, expressing the gene fusion in host cells transformedwith the recombinant expression vector, and allowing the expressedfusion protein to assemble much like antibody molecules, whereuponinterchain disulfide bonds form between the Fc moieties to yield thedimer.

The term “Fc polypeptide” includes native and mutein forms ofpolypeptides derived from the Fc region of an antibody. Truncated formsof such polypeptides containing the hinge region that promotesdimerization also are included. Fusion proteins comprising Fc moieties(and oligomers formed therefrom) offer the advantage of facilepurification by affinity chromatography over Protein A or Protein Gcolumns.

Another method for preparing oligomeric antigen binding proteinsinvolves use of a leucine zipper. Leucine zipper domains are peptidesthat promote oligomerization of the proteins in which they are found.Leucine zippers were originally identified in several DNA-bindingproteins (Landschulz et al., 1988, Science 240:1759), and have sincebeen found in a variety of different proteins. Among the known leucinezippers are naturally occurring peptides and derivatives thereof thatdimerize or trimerize. Examples of leucine zipper domains suitable forproducing soluble oligomeric proteins are described in WO 94/10308, andthe leucine zipper derived from lung surfactant protein D (SPD)described in Hoppe et al., 1994, FEBS Letters 344:191. The use of amodified leucine zipper that allows for stable trimerization of aheterologous protein fused thereto is described in Fanslow et al., 1994,Semin. Immunol. 6:267-78. In one approach, recombinant fusion proteinscomprising an anti-AIP2 antibody fragment or derivative fused to aleucine zipper peptide are expressed in suitable host cells, and thesoluble oligomeric anti-AIP2 antibody fragments or derivatives that formare recovered from the culture supernatant.

Antigen-binding fragments of antigen binding proteins of the inventionmay be produced by conventional techniques. Examples of such fragmentsinclude, but are not limited to, Fab and F(ab′)₂ fragments.

The present disclosure provides monoclonal antibodies that bind to AIP2.Monoclonal antibodies may be produced using any technique known in theart, e.g., by immortalizing spleen cells harvested from the transgenicanimal after completion of the immunization schedule. The spleen cellscan be immortalized using any technique known in the art, e.g., byfusing them with myeloma cells to produce hybridomas. Myeloma cells foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency, and enzymedeficiencies that render them incapable of growing in certain selectivemedia which support the growth of only the desired fused cells(hybridomas). Examples of suitable cell lines for use in mouse fusionsinclude Sp-20, P3-X63/Ag8, P3-X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14, FO,NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; examples of celllines used in rat fusions include R210.RCY3, Y3-Ag 1.2.3, IR983F and48210. Other cell lines useful for cell fusions are U-266, GM1500-GRG2,LICR-LON-HMy2 and UC729-6.

Antigen binding proteins directed against AIP2 can be used, for example,in assays to detect the presence of AIP2 polypeptides, either in vitroor in vivo. The antigen binding proteins also may be employed inpurifying AIP2 proteins by immunoaffinity chromatography. Blockingantigen binding proteins can be used in the methods disclosed herein.Such antigen binding proteins that function as AIP2 antagonists may beemployed in treating any AIP2-induced condition, including but notlimited to various cancers.

Antigen binding proteins may be employed in an in vitro procedure, oradministered in vivo to inhibit AIP2-induced biological activity.Disorders caused or exacerbated (directly or indirectly) by theproteolytic activation of AIP2, examples of which are provided herein,thus may be treated. In one embodiment, the present invention provides atherapeutic method comprising in vivo administration of a AIP2 blockingantigen binding protein to a mammal in need thereof in an amounteffective for reducing an AIP2-induced biological activity.

Antigen binding proteins include fully human monoclonal antibodies thatinhibit a biological activity of AIP2.

Antigen binding proteins may be prepared by any of a number ofconventional techniques. For example, they may be purified from cellsthat naturally express them (e.g., an antibody can be purified from ahybridoma that produces it), or produced in recombinant expressionsystems, using any technique known in the art. See, for example,Monoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Kennet et al. (eds.), Plenum Press, New York (1980); andAntibodies: A Laboratory Manual, Harlow and Land (eds.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1988).

Any expression system known in the art can be used to make therecombinant polypeptides of the invention. In general, host cells aretransformed with a recombinant expression vector that comprises DNAencoding a desired polypeptide. Among the host cells that may beemployed are prokaryotes, yeast or higher eukaryotic cells. Prokaryotesinclude gram negative or gram positive organisms, for example E. coli orbacilli. Higher eukaryotic cells include insect cells and establishedcell lines of mammalian origin. Examples of suitable mammalian host celllines include the COS-7 line of monkey kidney cells (ATCC CRL 1651)(Gluzman et al., 1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK(ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived from theAfrican green monkey kidney cell line CV1 (ATCC CCL 70) as described byMcMahan et al., 1991, EMBO J. 10: 2821. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described by Pouwels et al. (Cloning Vectors: ALaboratory Manual, Elsevier, N.Y., 1985).

The transformed cells can be cultured under conditions that promoteexpression of the polypeptide, and the polypeptide recovered byconventional protein purification procedures. One such purificationprocedure includes the use of affinity chromatography, e.g., over amatrix having all or a portion (e.g., the extracellular domain) of AIP2bound thereto. Polypeptides contemplated for use herein includesubstantially homogeneous recombinant mammalian anti-AIP2 antibodypolypeptides substantially free of contaminating endogenous materials.

Antigen binding proteins may be prepared, and screened for desiredproperties, by any of a number of known techniques. Certain of thetechniques involve isolating a nucleic acid encoding a polypeptide chain(or portion thereof) of an antigen binding protein of interest (e.g., ananti-AIP2 antibody), and manipulating the nucleic acid throughrecombinant DNA technology. The nucleic acid may be fused to anothernucleic acid of interest, or altered (e.g., by mutagenesis or otherconventional techniques) to add, delete, or substitute one or more aminoacid residues, for example.

Single chain antibodies may be formed by linking heavy and light chainvariable domain (Fv region) fragments via an amino acid bridge (shortpeptide linker), resulting in a single polypeptide chain. Suchsingle-chain Fvs (scFvs) have been prepared by fusing DNA encoding apeptide linker between DNAs encoding the two variable domainpolypeptides (V_(L) and V_(H)). The resulting polypeptides can fold backon themselves to form antigen-binding monomers, or they can formmultimers (e.g., dimers, trimers, or tetramers), depending on the lengthof a flexible linker between the two variable domains (Kortt et al.,1997, Prot. Eng. 10:423; Kortt et al., 2001, Biomol. Eng. 18:95-108). Bycombining different V_(L) and V_(H)-comprising polypeptides, one canform multimeric scFvs that bind to different epitopes (Kriangkum et al.,2001, Biomol. Eng. 18:31-40). Techniques developed for the production ofsingle chain antibodies include those described in U.S. Pat. No.4,946,778; Bird, 1988, Science 242:423; Huston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879; Ward et al., 1989, Nature 334:544, de Graaf etal., 2002, Methods Mol. Biol. 178:379-87.

Techniques are known for deriving an antibody of a different subclass orisotype from an antibody of interest, i.e., subclass switching. Thus,IgG antibodies may be derived from an IgM antibody, for example, andvice versa. Such techniques allow the preparation of new antibodies thatpossess the antigen-binding properties of a given antibody (the parentantibody), but also exhibit biological properties associated with anantibody isotype or subclass different from that of the parent antibody.Recombinant DNA techniques may be employed. Cloned DNA encodingparticular antibody polypeptides may be employed in such procedures,e.g., DNA encoding the constant domain of an antibody of the desiredisotype (Lantto et al., 2002, Methods Mol. Biol. 178:303-16). Moreover,if an IgG4 is desired, it may also be desired to introduce a pointmutation (CPSCP→CPPCP) in the hinge region (Bloom et al., 1997, ProteinScience 6:407) to alleviate a tendency to form intra-H chain disulfidebonds that can lead to heterogeneity in the IgG4 antibodies.

In particular embodiments, antigen binding proteins of the presentinvention have a binding affinity (K_(a)) for AIP2 of at least 10⁶. Inother embodiments, the antigen binding proteins exhibit a K_(a) of atleast 10⁷, at least 10⁸, at least 10⁹, or at least 10¹⁰. In anotherembodiment, the antigen binding protein exhibits a K_(a) substantiallythe same as that of an antibody described herein in the Examples.

In another embodiment, the present disclosure provides an antigenbinding protein that has a low dissociation rate from AIP2. In oneembodiment, the antigen binding protein has a K_(off) of 1×10⁻⁴ to ⁻¹ orlower. In another embodiment, the K_(off) is 5×10⁻⁵ to ⁻¹ or lower. Inanother embodiment, the K_(off) is substantially the same as an antibodydescribed herein. In another embodiment, the antigen binding proteinbinds to AIP2 with substantially the same K_(off) as an antibodydescribed herein.

In another aspect, the present disclosure provides an antigen bindingprotein that inhibits an activity of AIP2. In one embodiment, theantigen binding protein has an IC₅₀ of 1000 nM or lower. In anotherembodiment, the IC₅₀ is 100 nM or lower; in another embodiment, the IC₅₀is 10 nM or lower. In another embodiment, the IC₅₀ is substantially thesame as that of an antibody described herein in the Examples. In anotherembodiment, the antigen binding protein inhibits an activity of AIP2with substantially the same IC₅₀ as an antibody described herein.

In another aspect, the present disclosure provides an antigen bindingprotein that binds to human AIP2 expressed on the surface of a cell and,when so bound, inhibits AIP2 signaling activity in the cell withoutcausing a significant reduction in the amount of AIP2 on the surface ofthe cell. Any method for determining or estimating the amount of AIP2 onthe surface and/or in the interior of the cell can be used. In otherembodiments, binding of the antigen binding protein to theAIP2-expressing cell causes less than about 75%, 50%, 40%, 30%, 20%,15%, 10%, 5%, 1%, or 0.1% of the cell-surface AIP2 to be internalized.

In another aspect, the present disclosure provides an antigen bindingprotein having a half-life of at least one day in vitro or in vivo(e.g., when administered to a human subject). In one embodiment, theantigen binding protein has a half-life of at least three days. Inanother embodiment, the antigen binding protein has a half-life of fourdays or longer. In another embodiment, the antigen binding protein has ahalf-life of eight days or longer. In another embodiment, the antigenbinding protein is derivatized or modified such that it has a longerhalf-life as compared to the underivatized or unmodified antigen bindingprotein. In another embodiment, the antigen binding protein contains oneor more point mutations to increase serum half life, such as describedin WO00/09560, incorporated by reference herein.

The present disclosure further provides multi-specific antigen bindingproteins, for example, bispecific antigen binding protein, e.g., antigenbinding protein that bind to two different epitopes of AIP2, or to anepitope of AIP2 and an epitope of another molecule, via two differentantigen binding sites or regions. Moreover, bispecific antigen bindingprotein as disclosed herein can comprise an AIP2 binding site from oneof the herein-described antibodies and a second AIP2 binding region fromanother of the herein-described antibodies, including those describedherein by reference to other publications. Alternatively, a bispecificantigen binding protein may comprise an antigen binding site from one ofthe herein described antibodies and a second antigen binding site fromanother AIP2 antibody that is known in the art, or from an antibody thatis prepared by known methods or the methods described herein.

Numerous methods of preparing bispecific antibodies are known in theart. Such methods include the use of hybrid-hybridomas as described byMilstein et al., 1983, Nature 305:537, and chemical coupling of antibodyfragments (Brennan et al., 1985, Science 229:81; Glennie et al., 1987,J. Immunol. 139:2367; U.S. Pat. No. 6,010,902). Moreover, bispecificantibodies can be produced via recombinant means, for example by usingleucine zipper moieties (i.e., from the Fos and Jun proteins, whichpreferentially form heterodimers; Kostelny et al., 1992, J. Immunol.148:1547) or other lock and key interactive domain structures asdescribed in U.S. Pat. No. 5,582,996. Additional useful techniquesinclude those described in U.S. Pat. Nos. 5,959,083; and 5,807,706.

In another aspect, the antigen binding protein comprises a derivative ofan antibody. The derivatized antibody can comprise any molecule orsubstance that imparts a desired property to the antibody, such asincreased half-life in a particular use. The derivatized antibody cancomprise, for example, a detectable (or labeling) moiety (e.g., aradioactive, colorimetric, antigenic or enzymatic molecule, a detectablebead (such as a magnetic or electrodense (e.g., gold) bead), or amolecule that binds to another molecule (e.g., biotin or streptavidin),a therapeutic or diagnostic moiety (e.g., a radioactive, cytotoxic, orpharmaceutically active moiety), or a molecule that increases thesuitability of the antibody for a particular use (e.g., administrationto a subject, such as a human subject, or other in vivo or in vitrouses). Examples of molecules that can be used to derivatize an antibodyinclude albumin (e.g., human serum albumin) and polyethylene glycol(PEG). Albumin-linked and PEGylated derivatives of antibodies can beprepared using techniques well known in the art. In one embodiment, theantibody is conjugated or otherwise linked to transthyretin (TTR) or aTTR variant. The TTR or TTR variant can be chemically modified with, forexample, a chemical selected from the group consisting of dextran,poly(n-vinyl pyrrolidone), polyethylene glycols, propropylene glycolhomopolymers, polypropylene oxide/ethylene oxide co-polymers,polyoxyethylated polyols and polyvinyl alcohols.

Example 1

Antibodies were immobilized on CM5 sensor chip using standard NHS/EDCcoupling methodology. All measurements were conducted in HBS-EP bufferwith a flow rate of 30 μL/min. AIP2 was diluted so as to obtain a seriesof concentrations. A 1:1 (Langmuir) binding model was

TABLE 1 mAb K_(on) K_(off) K_(d) C7 3.08E+5 0.016 52 nM D3 5.22E+50.0254 49 nM E7 1.15E+6 0.0736 64 nMused to fit the data.

Example 2

This example shows quorum quenching using S. aureus agr-driven YFPreporter strains. S. aureus strains (transformed with the reporterplasmid pDB59) representing agr group I (USA300/LAC), agr group II(USA100), and agr group IV (MnTG). All S. aureus reporter strains weregrown overnight in tryptic soy broth supplemented with chloramphenicolat 10 μg/mL at 37° C. with shaking. For antibody inhibition testing,cultures were diluted 100-fold into fresh media, incubated for 1 hour at37° C., and 180 μL was dispensed into wells of a 96-well microtiterplate. The anti-AIP 2 mAbs were diluted to a working concentration of 3mg/mL in PBS, and 3-fold serial dilutions were generated in PBS to afinal concentration of 4.6×10⁻⁴ mg/mL. 20 μL of each antibody dilutionwas added to the reporter cultures in the microtiter plates intriplicate, resulting in an additional 10-fold dilution. As controls,PBS was used as a mock treated sample (no antibody). Plates wereincubated for 22 h at 37° C. with shaking at 250 rpm. Absorbance andfluorescence was measured in a plate reader using an excitationwavelength of 485 nm and an emission wavelength of 530 nm.

TABLE 2 Inhibition Inhibition Inhibition of AIP1 QS of AIP2 QS of AIP4QS mAb (IC₅₀; nM) (IC₅₀; nM) (IC₅₀; nM) C7 109.9 33.7 (USA100 - AH2619)86    (USA300 - LAC) 31.2 (USA100 - AH2625) (MnTG) E7  91.1 34.2(USA100 - AH2619) 53.5 (USA300 - LAC) 31.02 (USA100 - AH2625)  (MnTG)

Example 3

This example shows mitigation of a MRSA lethal peritonitis infectionmodel. The S. aureus strain USA100 was harvested at mid-exponentialgrowth phase washed in sterile Dulbecco's phosphate buffered saline(DPBS) and resuspended in sterile DPBS. To test the efficacy of theanti-AIP2 antibodies against a lethal challenge with S. aureus USA100,mice were pretreated for one-hour with 1 mg of antibody viaintraperitoneal (i.p.) inoculation. After pre-treatment all mice wereinfected i.p. with 100 μL containing 5.5×10⁷ colony forming units (CFUs)for infections with USA100. Mice were monitored every 3 h for the first48 h and then twice per day for 72 hours. If the animals were immobileor unable to eat or drink, they were euthanized. Pre-treatment with bothC7 and E7 resulted in decreases in lethality (FIG. 1). Remarkably, micepre-treated with E7 had an 80% survival rate compared to 0 and 10%survival in control IgG and PBS control groups.

Example 4

This example shows mitigation of a MRSA skin and soft tissue infection(SSTI) in a mouse model. For the SSTI model, BALB/c mice (8 mice pertreatment group) were shaved and infected subcutaneously with 2×10⁶USA100 mixed with 1 mg of C7, E7, control mAb, or PBS. Infected area wasmeasured daily for 10 days. Treatment with E7 caused significantreduction in abscess size at all days tested (FIG. 2).

Example 5

This example shows mitigation of a MSSA lethal peritonitis infectionmodel. The S. aureus strain RN4850, which produced AIP4, was harvestedat mid-exponential growth phase washed in sterile Dulbecco's phosphatebuffered saline (DPBS) and resuspended in sterile DPBS. To test theefficacy of the anti-AIP2 antibodies against a lethal challenge with anAIP4-producing S. aureus RN4850, mice were pretreated for one-hour with1 mg of antibody via intraperitoneal (i.p.) inoculation. Afterpre-treatment all mice were infected i.p. with 100 μL containing 2×10⁸colony forming units (CFUs) for infections with RN4850. Mice weremonitored every 3 h for the first 48 h and then twice per day for 72hours. If the animals were immobile or unable to eat or drink, they wereeuthanized. Pre-treatment with E7 resulted in decreases in lethality(FIG. 3). Remarkably, mice pre-treated with E7 had a 50% survival ratecompared to 10% and 0% survival in control IgG and PBS control groups,respectively. These data demonstrate that the anti-AIP2 mAb E7 conferscross-protection against an AIP4-producing S. aureus strain.

We claim:
 1. A fully human antibody of an IgG class that binds to S. aureus auto inducing peptide-2 (AIP2) with a binding affinity of at least 10⁻⁶ M, wherein the antibody has a heavy chain/light chain variable domain sequence that is at least 95% identical to the amino acid sequences selected from the group consisting of SEQ ID NO. 3/SEQ ID NO. 4 (called C7 herein), SEQ ID NO. 5/SEQ ID NO. 6 (called D3 herein), SEQ ID NO. 7/SEQ ID NO. 8 (called E7 herein).
 2. A Fab fully human antibody fragment that binds to S. aureus auto inducing peptide-2 (AIP2) with a binding affinity of at least 10⁻⁶ M, wherein the antibody has a heavy chain/light chain variable domain sequence that is at least 95% identical to the amino acid sequences selected from the croup consisting of SEQ ID NO. 3/SEQ ID NO. 4 (called C7 herein), SEQ ID NO. 5/SEQ ID NO. 6 (called D3 herein), SEQ ID NO. 7/SEQ ID NO. 8 (called E7 herein).
 3. A single chain human antibody that binds to S. aureus auto inducing peptide-2 (AIP2) with a binding affinity of at least 10⁻⁶ M, having a variable domain region from a heavy chain and a variable domain region from a light chain and a peptide linker connection the heavy chain and light chain variable domain regions, wherein the antibody has a heavy chain/light chain variable domain sequence that is at least 95% identical to the amino acid sequences selected from the croup consisting of SEQ ID NO. 3/SEQ ID NO. 4 (called C7 herein), SEQ ID NO. 5/SEQ ID NO. 6 (called D3 herein), SEQ ID NO. 7/SEQ ID NO. 8 (called E7 herein).
 4. A method for treating a S. aureus infection, comprising administering an effective amount of an anti-AIP2 polypeptide, wherein the anti-AIP2 polypeptide is selected from the group consisting of a fully human antibody of an IgG class that binds to S. aureus auto inducing peptide-2 (AIP2) with a binding affinity of at least 10^(−6 M), wherein the antibody has a heavy chain/light chain variable domain sequence that is at least 95% identical to the amino acid sequences selected from the croup consisting of SEQ ID NO. 3/SEQ ID NO. 4 (called C7 herein), SEQ ID NO. 5/SEQ ID NO. 6 (called D3 herein), SEQ ID NO. 7/SEQ ID NO. 8 (called E7 herein); a Fab fully human antibody fragment, wherein the antibody has a heavy chain/light chain variable domain sequence that is at least 95% identical to the amino acid sequences selected from the group consisting of SEQ ID NO. 3/SEQ ID NO. 4 (called C7 herein), SEQ ID NO. 5/SEQ ID NO. 6 (called D3 herein), SEQ ID NO. 7/SEQ ID NO. 8 (called E7 herein); and a single chain human antibody, having a variable domain region from a heavy chain and a variable domain region from a light chain and a peptide linker connection the heavy chain and light chain variable domain regions, wherein the antibody has a heavy chain/light chain variable domain sequence that is at least 95% identical to the amino acid sequences selected from the group consisting of SEQ ID NO. 3/SEQ ID NO. 4 (called C7 herein), SEQ ID NO. 5/SEQ ID NO. 6 (called D3 herein), SEQ ID NO. 7/SEQ ID NO. 8 (called E7 herein).
 5. The method for treating a S. aureus infection of claim 4, wherein the S. aureus infection is caused by MRSA. 